Interest has been growing in protein conjugates formed by chemically linking two or more proteins. For example, cytotoxins may be linked to a monoclonal antibody specific for cells of diseased tissue or tumors. The monoclonal antibody seeks out the undesired cell and the cytotoxin destroys it without affecting normal and healthy cells. The commercial value of such conjugates was discussed in an article on page D6 of the New York Times of Jul. 1, 1987 in an article entitled "Aiming `Bullets` At Disease".
A conjugate can include two proteins, or more than two proteins. An example of a conjugate having many protein units was reported by Poznansky et al. in Science, 223, 1304-1306 (1984). The conjugate described by Poznansky, et al. contained the enzyme 1,4-glucosidase, albumin and insulin in a ratio of 1:10:60, and reportedly had an average molecular weight of 1.1.times.10.sup.6. The purpose of the conjugate was an improved treatment of patients suffering from low glycogen levels in muscle cells of cardiac and respiratory tissue due to type 2 glycogenesis. Conjugating the enzyme to albumin was reported to have rendered the enzyme both non-antigenic and non-immunogenic.
Polyproteins having a different purpose were disclosed by Ward, et al., U.S. Pat. No. 4,687,732. The Ward, et al. patent discloses visualization polymers linked to detecting agents. The visualization polymers contain proteins capable of being visualized when bound with a substrate. The detecting agent is specific for a target molecule that is the subject of an assay. For example, if the molecule being assayed is an antigen, the detecting agent may be an antibody. The multiple units of the visualization polymer amplify the signal that would be expected from binding the detecting agent to the target molecule.
Protein conjugates are prepared by linking proteins together using bifunctional reagents. The bifunctional reagents can be homobifunctional or heterobifunctional.
Homobifunctional reagents are molecules with at least two identical functional groups. The functional groups of the reagent generally react with one of the functional groups on a protein, typically an amino group.
Examples of homobifunctional reagents include glutaraldehyde and diimidates. An example of the use of glutaraldehyde as a cross-linking agent was the preparation of the enzyme-albumin-insulin polymer by Poznansky as described above. (See also Poznansky et al, Science, 223, 1304-1306 (1984)). The use of diimidates as a cross linking agent was described by Wang, et al. in Biochemistry, 16, 2937-2941 (1977).
Heterobifunctional regents have at least two different functional groups, each of which is capable of reacting with a different functional group of a protein. The different functional groups of a protein are typically an amino group and a thiol group.
The heterobifunctional reagents provide a more sophisticated method for linking two proteins. An example of a heterobifunctional reagent is N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). SPDP was described by Carlsson et al. in Biochem. J., 173, 723-737 (1978). The linking of two proteins, arbitrarily referred to as protein-1 and protein-2, is provided in scheme 1.
Another heterobifunctional reagent for linking proteins is succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), which is described by Yoshitake et al., Eur. J. Biochem, 101, 395-399 (1979). The use of SMCC to form a conjugate of two proteins may be described as shown in Scheme 2.
A number of additional heterobifunctional reagents for forming conjugates of two proteins is described by Rodwell et al., U.S. Pat. No. 4,671,958. Strategies for designing antibody-conjugates are reviewed by Ahmad et al., Trends in Biotechnology, 6, 246-251 (1988).
The disadvantage of the homobifunctional reagents is the random attachment of different proteins. In attempting to link a first protein with a second protein by means of a homobifunctional reagent, one cannot prevent the linking of the first protein to each other and of the second to each other.
The disadvantage of homobifunctional reagents is overcome by means of the heterobifunctional reagents. With the heterobifunctional reagents, one can control the sequence of reactions, and combine proteins at will.
Suggestions have been made to extend the use of heterobifunctional reagents beyond the preparation of protein conjugates containing two proteins. Thus, Carlsson et al., who disclose the use of SPDP to link proteins (see above) suggest at pages 733 and 734 of their article the possibility of producing tri- and tetra-protein conjugates by increasing the number of heterobifunctional reagents in the proteins. No experimental details of how to do so, however, are provided.
Similarly, Ward et al., U.S. Pat. No. 4,687,732, which discloses polyproteins for visualizing target molecules (see above), suggests the possibility of using heterobifunctional reagents for preparing the polymers. No experimental details are given, however. Only the homobifunctional reagent disuccinimidyl suberate is exemplified for linking proteins into the visualization polymer. See column 23, line 67 et sec. of U.S. Pat. No. 4,687,732 and Leary, et al., Proc. Natl. Acad. Sci. USA, 80. 4045-4049 (1983).
The ability to carry out rational schemes for producing polymers wherein the monomer units are proteins is desirable. It is not enough, however, merely to state that heterobifunctional reagents can be used to prepare polymers of proteins in the same way they are used to prepare dimers. There is a need, therefore, for a method of producing conjugates comprising polymers of proteins using heterobifunctional reagents to link the proteins together. It is an object of the present invention to provide such a method. It is a further object of the invention to prepare such polyproteins.